Ion channels come in a variety of forms, each fine-tuned by evolution to perform a particular function in a cell. We focus our attention on the gating properties of cyclic nucleotide-gated (CNG) channels, a family of ion channels that play key roles in the transduction of visual and olfactory information (Stryer, 1986, 1988; Zufall et al., 1994). These channels sense the variations in the intracellular concentration of cyclic nucleotides that occur in response to either visual or olfactory stimuli. In many ways, CNG channels are similar to voltage-activated potassium (KV) channels. They are tetramers with each subunit containing 6 transmembrane segments and a P-region between transmembrane segments S5 and S6. The main differences are that CNG channels poorly select among cations and they are not gated by changes in membrane voltage. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP. Sensitivity to cyclic nucleotides is conferred by the presence of a cyclic nucleotide binding domain at the C-terminus of each subunit. Our understanding of how CNG channels open and close their pore is less refined than in KV channels. Where is the gate in CNG channels? Some evidence suggests that the activation gate in CNG channels might not be at the intracellular side of the S6 transmembrane segment, as we found in KV channels (Liu et al., 1997; del Camino and Yellen, 2001). For example, tetracaine appears to block the permeation pathway of the channel, however, this blocker prefers the closed channel (Fodor et al., 1997a; Fodor et al., 1997b). In addition, experiments examining the state-dependence of cysteine modification by intracellular application MTS reagents or Ag+ ions failed to show dramatic differences between open and closed states in the inner vestibule region (Sun et al., 1996; Flynn and Zagotta, 2001), in contrast to observations in KV channels (Liu et al., 1997; del Camino and Yellen, 2001). What structural differences between KV and CNG channels might account for these disparate observations? Could it be that the closed state of CNG channels has a higher conductance than we measured for KV channels? Might the gates in these two related cation channels be located in very different regions? With the combined use of blockers, cysteine substitution, chemical modification and the ability of the blocker to protect modification, we are addressing these fundamental questions. The use of quaternary ammonium (QA) derivatives as a tool to understand protein function has been successfully used in the K+ channel field (Armstrong, 1966, 1969; Armstrong, 1971; Armstrong and Hille, 1972; Choi et al., 1993; Holmgren et al., 1997). These studies revealed that the gate is located at the intracellular side of the channel and that a cavity large enough to accommodate a molecule 8 in diameter is positioned above the gate. All these ideas were confirmed by the crystal structures of KcsA, a related K+ channel from bacteria (Doyle et al., 1998; Zhou et al., 2001a) and KV channels (Long et al., 2005). We have embarked on a detailed study of the interactions between QA compounds and CNG channels. We showed that QA compounds bind to CNG channels in both open and closed states, in stark contrast with the blockade mechanism in KV channels. Therefore, we concluded, as our colleagues have done previously (Sun et al., 1996; Flynn and Zagotta, 2001), that the S6 does not function as an activation gate in CNG channels. Where is the gate located? Even though the selectivity filter has been proposed to be the primary gate in CNG channels, there is little direct and clear evidence to support this idea. A systematic cysteine substitution study of the entire P region as target for chemical modification with extracellular MTS reagents revealed that the pore helix undergoes conformational changes associated with gating (Liu and Siegelbaum, 2000). The pore helix, however, does not line the permeation pathway in any of the cation channel X-ray structures, and thus a consistent state dependent pattern along residues lining the permeation pathway of CNG channels remains to be established. Recently, the crystal structure of a bacterial non-selective cation channel has been solved (Shi et al., 2006) and the structure of the P-region of this channel has been proposed to be equivalent to that of CNG channels. Using this structure as a model, we have introduced cysteines along the selectivity filter as targets for chemical modification with small cysteine reagents like Cd2+ and Ag+ ions applied from the intracellular side of the channel. If the selectivity filter is indeed the primary gate in CNG channels, we expect to observe a state dependent pattern of chemical modification consistent with gating, where some positions are accessible in open and closed states, while others are only accessible in open states, similar to our observations in KV channels (Liu et al., 1997).